The invention relates to affinity assays for the detection of a biological ligand such as a protein, particularly an antibody, or a nucleic acid. In particular, the invention includes the efficient detection of bifunctional biological ligands, such an antibody in whole blood and in fluids of animals, plants and other organisms, or of DNA labeled with two or more biological ligands. More particularly, the invention relates to an affinity assay in which the binding of such a ligand to a ligand receptor results in an electrochemical signal, such as a current or a potential.
Affinity assay systems are commonly used in clinical and non-clinical situations to detect, monitor, or confirm the identity, amount, or presence of a particular ligand. Examples include immunoassays for the detection of an antibody or antigen, such as enzyme linked immunoassay (ELISA) or radioimmunoassay (RIA). Such affinity assays confer specificity and sensitivity to the analysis of a particular ligand in a complex sample, such as blood or other body fluid.
Conventional affinity assays employing conventional labeling and detection techniques typically require washing and/or separation steps. In addition, many affinity assay systems require detection in a machine such as a spectrophotometer or fluorimeter. These are not practical when detection of the ligand in whole blood and other strongly light absorbing or scattering biological fluids is desired. Some conventional affinity assays, not requiring such equipment, rely on visible color changes for detection of ligand, which is also not practical in an opaque or colored fluid such as blood. Furthermore, the commonly used detection compounds, of conventional assays, such as hydrogen peroxide, are rapidly eliminated by protective enzymes found in blood and other tissues, such as catalase.
An affinity assay providing sensitive, efficient, and rapid detection of a ligand in a complex sample medium, and particularly for detection in whole blood is needed. A preferred assay would not require washing or separation steps, sample removal to machinery for analysis, and most preferably, would utilize only materials contained or generated in its probe, materials available in the biological fluids analyzed or, if added, not rapidly decomposed by enzymes in biological fluids. An affinity assay satisfying these criteria would permit the production of affinity assay systems to detect ligands in whole blood. Such an affinity assay system is described in the instant invention.
The affinity assay system of the present invention is based on the electrical connection of the third member, upon its binding to the second member, when the second member is located on or in the redox polymer film on the detecting electrode. The connection of the detection marker to the electrode is via the conducting redox polymer. The affinity assay system of the invention is capable of detecting and/or quantitating a variety of specific ligands, including proteins and nucleic acids, without washing or separation steps. The affinity assay system of the invention operates in whole blood and in other unseparated biological fluids, such as those of tissues and living cell cultures, without added toxic or unstable agents.
The affinity assay system of the invention includes an electrode coated with a conducting redox polymer, preferably a redox hydrogel. The redox polymer has multiple fast redox centers. The system has at least three members. The first and second members and also the second and third members are capable of conjugating with each other, and therefore capable of binding with each other. The first member of the ligandxe2x80x94ligand receptor pair is immobilized within the redox polymer either through an affinity reaction or by covalent bonding. The second member of the ligandxe2x80x94ligand receptor pair binds to the first. The third member is labeled with an amplifying detection marker, such as a peroxidase. Generation of the detection compound, the substrate of the detection maker, is catalyzed by another enzyme, such as choline oxidase. The enzyme that catalyzes the generation of a detection compound is immobilized in the redox polymer, but its reaction centers are preferably not oxidized by oxidized redox centers of the polymer and are not reduced by reduced redox centers of the polymer when the electrode is poised at its operating potential.
In a preferred embodiment, the affinity assay system includes an electrode coated with an electron conducting redox polymer in which a strongly binding member of a bioconjugating couple, such as ss-DNA or ss-peptide DNA, avidin, or streptoavidin and a substrate generating enzyme, such as hydrogen peroxide-generating choline oxidase, are immobilized. (xe2x80x9cssxe2x80x9d means single-stranded.) The first member of a ligandxe2x80x94ligand receptor pair is biotinylated or labeled with DNA or peptide DNA, and bound to the redox polymer via DNA hybridization or avidin-biotin coupling. The second member is bound then to the first. The third member is labeled with the detection marker horseradish peroxidase or soybean peroxidase. Binding of the labeled third member to the electrode via the second member results in electrical contact between the peroxidase and the redox polymer, causing the electrical connection of the reaction centers of the peroxidase label to the electrode through the conducting redox polymer. Such connection converts the film to a catalyst for the electroreduction of the hydrogen peroxide produced within the film by the immobilized substrate-generating enzyme.